1. Field of the Invention
The present invention relates to protein study and more particularly to methods and devices for crystallizing proteins suitable for x-ray diffraction studies.
2. Description of Related Art
The conformational structure of proteins is a key to understanding their biological functions and to ultimately designing new drug therapies. The conformational structures of proteins are conventionally determined by x-ray diffraction from their crystals. Unfortunately, growing protein crystals of sufficient high quality is very difficult in most cases, and such difficulty is the main limiting factor in the scientific determination and identification of the structures of protein samples. Prior art methods for growing protein crystals from super-saturated solutions are tedious and time-consuming, and less than two percent of the over 100,000 different proteins have been grown as crystals suitable for x-ray diffraction studies.
Missing protein structural information is a significant obstacle to the rational design of drugs and vaccines for diseases such as AIDS. Of the proteins encoded by the AIDS genome, only one, namely HIV protease, has a known three-dimensional structure. The remaining proteins, in particular the HIV reverse transcriptase molecule which is essential for retroviral replication, have so far proven resistant to x-ray crystallography, which is, at present, the most widely-used technique for determining protein structures.
As another example, cystic fibrosis is an inherited disorder associated with a defective gene which codes for the protein known as cystic fibrosis transmembrane conductance regulator (CFTR). Approximately one person in thirty carries a defective gene for CFTR. If both spouses carry the gene, then one in fur of their children will be at severe risk for early death from pulmonary insufficiency and/or infection. Like the overwhelming majority of transmembrane proteins, CFTR is resistant to crystallization, and thus its structure cannot be determined by x-ray crystallography.
The problem in the prior art has been to determine the molecular structure of proteins to near atomic resolution, e.g., under two Angstroms. Many biologists require such resolution so that they can determine protein function. Such resolution indicates the use of x-rays, rather than electrons, to illuminate the proteins. Drug designers and protein engineers use the same conformational information to design adducts to dock with the protein and/or modify its function.
Protein structures of atomic resolution are catalogued by Brookhaven National Laboratory and made available to the public via the Internet, e.g., at "http://www.pdb.bnl.gov/". The Protein Data Bank (PDB) is an archival computer database of three-dimensional structures of biological macromolecules. The database contains atomic coordinates, bibliographic citations, primary sequence and secondary structure information, as well as crystallographic structure factors and 2D-NMR experimental data. Information is available on protein, DNA, RNA, virus and carbohydrate structures. The database is updated approximately every three weeks and a newsletter and CD-ROM are published quarterly. The data is normally distributed using the PDB interchange format which has become the standard for the field. The PDB is supported by a combination of Federal Government Agency funds and user fees. Support is provided by the U.S. National Science Foundation, the U.S. Public Health Service, National Institutes of Health, National Center for Research Resources, National Institutes of General Medical Sciences, and National Library of Medicine and the U.S. Department of Energy under contract DE-AC02-76Ch00016.
Brookhaven reports that progress in their endeavor of cataloguing 100,000 human proteins has been arduous. As of Jan. 17, 1996, there were 4,070 coordinate entries, 3,785 proteins, 273 nucleic acids, and 12 carbohydrates posted in the data bank.
One of the main reasons for this slow progress is the "protein crystallization bottleneck". Because proteins are small, they cannot be x-ray imaged individually. Instead, the many identical scattering centers found in a protein crystal are used. Then a reconstruction of their three-dimensional structure can be attempted by Fourier synthesis from an x-ray (or electron) diffraction pattern. The bottleneck in this process is in the tedious empirical lab work that growing protein crystals requires. Some protein crystals can take months to grow, some even take years. Many protein crystals prove to be completely elusive, or are of insufficient quality, no matter how patient a lab is in its experiments.
The application of electric fields to colloids has been used to change the optical properties of liquid crystals in a digital display. In electrophoresis, the electric field has been used to sort biomolecules. In complex fluids, a mixture of micron-sized particles in oil solidifies when exposed to an electric field, e.g., electrorheology which deals with the viscosity of fluids. Colloidal particles are observed to arrange themselves into a body-centered tetragonal lattice under the influence of the applied electric field. This lattice-on-demand effect can help protein crystallographers to produce diffraction quality crystals. The idea of aligning .alpha.-helical polypeptides in an electric field and letting them self-assemble into a two-dimensional crystal has already been demonstrated. See, Worley, et al., "Electric-Field-Enhanced Self-Assembly of .alpha.-Helixical Polypeptides", Langmuir, 11 (95) 3805.
Electrocrystallization of inorganics at an electrode is a well-known technique. However its application to proteins is not, albeit related to studies of protein electrode position and adsorption on electrode surfaces. But in electrorheology, insulation barriers are strategically placed so redox reactions and electrode currents are prevented, and thus such reactions and currents can be neglected.
Aled M. Edwards, et al., report in, "Epitaxial growth of protein crystals on lipid layers", Structural Biology, volume 1, number 3, March 1994, pp. 195-197, the growth of three-dimensional protein crystals seeded by two-dimensional crystals formed on lipid layers. Epitaxial growth was accomplished using glass coverslips coated with lipid layers of preformed 2-D crystals or with lipid layers alone, e.g., in a hanging drop.
Michael F. Toney, et al., describe in, "Voltage-dependent ordering of water molecules at an electrode-electrolyte interface", Nature, vol. 368, no. 31, March 1994, pp. 444, in situ x-ray scattering to investigate water distribution perpendicular to an aqueous-metal interface.
Other research indicates that crystal-like, ordered arrays of glass microspheres can be formed in seconds when subjected to a strong external electric field. Similar electric fields are available in electrochemical cells near the electrode interface.
In electrorheology, electric fields of 10.sup.6 V/m are used to migrate 20 .mu.m colloid particles from an isotropic fluid distribution to a body-centered tetragonal lattice of particles. In the liquid crystal art, comparable electric fields are used to switch smaller rod-like, or disk-like, particles from an isotropic distribution to a partially ordered nematic state. In electrochemistry, vicinal ice is known to form at the electrode-water interface where the field strength within the electric double layer can reach 10.sup.9 V/m. The physical mechanism in each of these cases is similar. It involves the rotational orientation of permanent and/or induced electric dipoles of particles by an applied electric field.
A field strength of 10.sup.8 V/m exceeds the bulk dielectric breakdown strength of water by two orders of magnitude. However, it is well known from fundamental studies of the electrochemical double layer that field strengths of this magnitude routinely exist near an electrode's surface. In other words, the voltage gradient between pairs of electrodes is not linear with distance. Most of the field is dropped within a few tens of Angstroms of the electrode surface. In the bulk, there is only a small electric field associated with the resistance of the solution. The bulk of an electrolyte merely feeds proteins to the double layer and promotes synthetic crystal growth.